1. Field of the Invention (Technical Field)
The present invention relates to solid and solution phase metallopeptide combinatorial libraries, metal ion-complexed peptidomimetic and peptide-like combinatorial libraries and metallo-construct combinatorial libraries, wherein at least a portion of each library constituent is conformationally constrained upon complexation with a metal ion, and methods for use and making of the same. The invention also relates to methods for synthesizing and assembling such libraries, and methods for identification and characterization of library constituents which are capable of binding a target molecule of interest, or mediating a biological activity of interest.
2. Background Art
Peptide Libraries and Combinatorial Chemistry. U.S. patent application Ser. No. 08/660,697 (the “'697 application”) teaches general combinatorial chemistry techniques for metallopeptides, including prior art methods now well recognized tools for rapid drug discovery. A library of peptides and other small molecules, with its enormous pool of structurally diverse molecules, is well suited for both lead generation as well as lead optimization. Libraries of a variety of molecular species have been described in literature and screened for drug discovery, including peptides, peptoids, peptidomimetics, oligonucleotides, benzodiazepines, and other libraries of small organic molecules.
Various approaches have been used to construct libraries of structurally diverse chemical compounds, include chemical synthesis and genetic engineering methods. Chemically synthesized libraries have been synthesized by general solution chemical means and by solid-phase methods. The prior art on designing, synthesizing, screening, and evaluation of peptide-based libraries has been reviewed in numerous articles, including the following, incorporated herein by reference: Pinilla C et al, Biopolymers (Peptide Sci) 37:221-240, 1995; Lebl M et al, Biopolymers (Peptide Sci) 37:177-198, 1995; Lam K S et al, Chemical Reviews 97:411-448, 1997; Smith G P and Petrenko G P, Chemical Reviews 97:391-410, 1997; Nefzi A et al, Chemical Reviews 97:449-472, 1997; Holmes C P et al, Biopolymers (Peptide Sci) 37:199-211, 1995; and, Moran E J et al, Biopolymers (Peptide Sci) 37:213-219, 1995.
Spatially Addressable Parallel Synthesis of Solid Phase Bound Libraries. Various strategies for chemical construction of a library of peptides or other small molecules are also well established. One strategy involves spatially separate synthesis of compounds in parallel on solid phase or on a solid surface in a predetermined fashion so that the location of one compound or a subset of compounds on the solid surface is known. The first such method was developed by Geysen for peptide epitope mapping (Geysen H M, Meloen R H, Barteling S J: Proc Natl Acad Sci USA 81:3998-4002, 1984). This method involves synthesis of various sets and subsets of a library of peptides on a multiple number of polypropylene pin tips in a predetermined fashion. The assembly of a library of greater than 10,000 molecules by this method is, however, cumbersome and time consuming. The light-directed spatially addressable parallel chemical synthesis technique (Fodor S P A et al: Science 251:767-773, 1991), based upon use of photolithographic techniques in peptide synthesis on a solid surface, such as a borosilicate glass microscope slide, is a better method of constructing libraries containing more than 100,000 spatially separated compounds in a pre-determined fashion. However, synthesis of libraries that are structurally more diverse than simple peptides requires the development of orthogonal photolabile protecting groups that can be cleaved at different wavelengths of light. In addition, the solid surface bearing these libraries also has been reported to cause a pronounced effect on binding affinities in library screening assays (Cho C Y et al: Science 261:1303-1305, 1993; Holmes C P et al: Biopolymers 37:199-211, 1995).
The DIVERSOMER® apparatus designed by DeWitt and coworkers at Parke-Davis Pharmaceutical Research Division of Warner-Lambert Company, Ann Arbor, Mich., USA, offers a convenient and automated method of parallel synthesis of small organic molecule libraries on a solid phase (DeWitt S H et al: Proc Natl Acad Sci USA 90:6909-6913, 1993; U.S. Pat. No. 5,324,483; DeWitt S H et al: Acc Chem Res 29:114-122, 1996). Another conceptually similar apparatus for the solid phase synthesis of small organic molecule libraries has been reported by Meyers and coworkers (Meyers H V et al: Molecular Diversity 1: 13-20, 1995).
Pooling and Split Synthesis Strategies. Large libraries of compounds can be assembled by a pooling strategy that employs equimolar mixtures of reactants in each synthetic step (Geysen H M et al: Mol Immunol 23:709-715, 1986) or preferably by adjusting the relative concentration of various reactants in the mixture according to their reactivities in each of the coupling reactions (Ostresh J M et al: Biopolymers 34:1681-1689, 1994; U.S. Pat. No. 5,010,175 to Rytter W J and Santi D V). In one approach equimolar mixtures of compounds are obtained by splitting the resin in equal portions, each of which is separately reacted with each of the various monomeric reagents. The resin is mixed, processed for the next coupling, and again split into equal portions for separate reaction with individual reagents. The process is repeated as required to obtain a library of desired oligomeric length and size. This approach is also the basis of the “one-bead one-peptide” strategy of Furka et al. and Lam et al. (Furka et al: Int. J. Peptide Protein Res. 37:487, 1991; Lam K S et al: Nature 354:82-84, 1991; Lam K S et al: Nature 360:768, 1992) which employs amino acid sequencing to ascertain the primary structure of the peptide on a hit bead in a bioassay. Automated systems have been developed for carrying out split synthesis of these libraries with rather more efficiency (Zukermann R N et al: Peptide Res 5:169-174, 1992; Zukermann R N et al: Int J Peptide Protein Res 40:497-506, 1992). A common artifact occasionally seen with all these resin bound libraries is altered target-specific affinity by some solid phase bound compounds in bioassays, which can result in totally misleading results.
Another strategy involves construction of soluble libraries (Houghten R A et al: Proc Natl Acad Sci USA 82:5131-5135, 1985; Berg et al: J Am Chem Soc 111:8024-8026, 1989; Dooley C T et al: Science 266:2019-2022, 1994; Blondelle S E: Antimicrob Agents Chemother 38:2280-2286, 1994; Panilla C: Biopolymers 37:221-240, 1995). This strategy involves a deconvolution process of iterative re-synthesis and bioassaying until all the initially randomized amino acid positions are defined. Several modifications to this strategy have been developed, including co-synthesis of two libraries containing orthogonal pools, as demonstrated by Tartar and coworkers, which eliminates the need of iterative re-synthesis and evaluation (Deprez B et al: J Am Chem Soc 117: 5405-5406, 1995). The positional scanning method devised by Houghton and coworkers eliminates iterative re-synthesis (Dooley C T et al: Life Sci 52:1509-1517, 1993; Pinilla C et al: Biotechniques 13:901-905, 1992; Pinilla C et al: Drug Dev Res 33:133-145, 1992). A combination of this strategy with the split synthesis methods described above has also been described (Erb E et al: Proc Natl Acad Sci USA, 91:11422-11426, 1994). A major limitation of the soluble library approach is its applicability to high affinity systems. The abundance of each compound in solution can be influenced by the total number of compounds in a library which can affect the biological activity. For this reason, a highly active compound in any pool may not in fact be the most potent molecule. Lack of reasonable solubilities of certain members in a library may further influence this phenomenon. In fact, for several libraries the most active peptide was not even identified in the most active library pool (Dooley C T et al: Life Sci 52:1509-1517, 1993; Eichler J, in Proc . 23rd Eur. Peptide Symp., Berga, September 1994, Poster 198; Wyatt J R: Proc Natl Acad Sci USA, 91:1356-1360, 1994).
Various strategies for determination of the structure for a positive hit in a random library have been developed. See, e.g., U.S. Pat. No. 5,698,301. For a solid-phase library, direct analytical modalities include Edman degradation for peptide libraries, DNA sequencing of oligonucleotide libraries, and various mass spectrometry techniques on matrix bound compounds. The technique of creating a series of partially end-capped compounds at each of the synthetic steps during library assembly helps their unambiguous identification by mass spectrometry (Youngquist R S et al: J Am Chem Soc 117:3900-3906, 1995; Youngquist R S et al: Rapid Commun Mass Spectr 8:77-81, 1994). Direct mass spectrometric analysis of compounds covalently bound to a solid phase matrix of particles is also now possible by the use of matrix-assisted laser desorption/ionization (MALDI) techniques (Siuzdak G et al: Bioorg Med Chem Lett 6:979, 1996; Brown B B et al: Molecular Diversity 1:4-12, 1995). In addition to these analytical techniques, various encoding strategies have been devised for structure elucidation in organic molecule-based libraries, including both non-peptide and non-nucleotide libraries, such as DNA encoding, peptide coding, haloaromatic tag encoding, and encoding based on radiofrequency transponders. See, e.g., U.S. Pat. No. 5,747,334.
Most of the libraries described above are termed “random” libraries because of their enormous structural and conformational diversity. Libraries of relatively restricted and biased structures have also been reported. Examples of libraries of conformationally rigid compounds built on a structurally common template include benzodiazepine, β-lactam, β-turn mimetics, diketopiperazines, isoquinolines, dihydro- and tetrahydroisoquinolines, 1,4 dihydropyridines, hydantoins, pyrrolidines, thiazolidine-4-carboxylic acids, 4-thiazolidines and related 4-metathiazanones and imidazoles.
Among the various classes of libraries of small molecules, peptide libraries remain the most versatile because of the structural diversity offered by the use of naturally occurring amino acids, incorporation of a variety of “designer” amino acids, and the high efficiency and ease with which peptide synthesis can be accomplished. In addition, another level of structural diversity in peptide-based libraries has been added by post-synthesis modification of the libraries. These modifications include permethylation, acylation, functionalization of the side chain functionality, and reductive amination of the N-terminus.
Many libraries specifically customized for one particular biological target have also been reported. These libraries are generally assembled by incorporating only a set of structural elements that might be essential for eliciting a target-specific response. Some of the reported libraries of this class include aspartic acid protease, zinc proteases, carbonic anhydrase inhibitors, tyrosine kinase inhibitors, estrogen receptor ligands, and antioxidants.